Nitric oxide synthase (NOS), a flavo-hemoprotein, tightly regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a key signaling molecule for vasodilatation and neurotransmission at low concentrations, and also as a defensive cytotoxin at higher concentrations. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their key biological functions by tight regulation of interdomain electron transfer (IET) process via interdomain interactions. Our long-term goal is to determine the molecular mechanism of NOS enzyme catalysis, including how the interdomain interactions modulate the catalytically relevant IET processes and the NOS enzyme function. In this project we will focus on investigating important roles of the FMN domain docking to the heme domain in regulating the NOS isoform function. An exciting recent development in the NOS enzymes is the discovery of importance of the interdomain FMN-heme interactions for modulating reactivity and structure of the catalytic heme active site. The recent findings raise two new important mechanistic questions that must be addressed to understand NOS regulation mechanism: 1) what specific interdomain interactions govern and facilitate the productive docking of the FMN domain to the heme domain? 2) how can the FMN domain docking, in addition to controlling the catalytically essential FMN-heme IET, modulate reactivity of the heme active site? Our two synergistic and complementary Aims are designed to directly address these important questions. We will employ an integrated program of pulsed electron paramagnetic resonance, laser flash photolysis and mutational studies. In the absence of a structure of full-length mammalian NOS, the combined approach is highly appropriate for the proposed study. This project seeks to address fundamental questions relating to the NOS enzymes. Our study will identify key structural determinants in controlling NOS isoform activity by modulating the alignment of the FMN and heme domains. The new information could facilitate bio-rational development of new selective activators or inhibitors for these clinically important enzymes, in order to provide better therapeutic interventions. In accordance with the guideline of the R15 program we will continue to train our 21st century biomedical scientists by this multi-disciplinary project.